Table 1.
Biomarkers and antibodies.
| Antigen | Clone | Species | Fixation | Use of Kit | wc (μg/mL) | Supplier | Cut-Off for Positivity |
|---|---|---|---|---|---|---|---|
| Primary antibodies | |||||||
| Integrin α2β1 | BHA2.1 | m | Acetone | - | 2.50 | Millipore, Burlington, MA, USA | ≥20% |
| ERα | 1D5 | m | Formalin | + | 2.50 | Dako, Santa Clara, CA, USA | ≥1% |
| PR | PgR 636 | m | Formalin | + | 2.50 | Dako, Santa Clara, CA, USA | ≥1% |
| HER-2/neu | 4B5 | r | Acetone | - | 1.50 | Ventana, Roche, Basel, CH | ≥10% (Intensity 2+/3+) |
| EGFR | H11 | m | Acetone | - | 2.94 | Dako, Santa Clara, CA, USA | ≥50% |
| HGFR | SP44 | r | Acetone | - | 2.12 | Spring Bioscience, Pleasanton, CA, USA | ≥50% |
| IGF1R | 23-41 | m | Acetone | + | 4.00 | invitrogen, Carlsbad, CA, USA | ≥80% |
| MUC-1 | Ma552 | m | Acetone | - | 0.50 | Monosan, Uden, NL | ≥70% |
| CD44v6 | VFF-18 | m | Acetone | - | 1.00 | affymetrix eBioscience, Santa Clara, CA, USA | ≥10% |
| Integrin αVβ3 | LM609 | m | Acetone | - | 5.00 | Millipore, Burlington, MA, USA | ≥20% |
| CD3 | UCHT1 | m | Acetone | - | 1.25 | BD Biosciences, Franklin Lakes, NJ, USA | |
| CD8 | C8/144B | m | Acetone | + | 3.00 | Dako, Santa Clara, CA, USA | |
| PD-1 | MIH4 | m | Acetone | + | 10.00 | affymetrix eBioscience, Santa Clara, CA, USA | |
| PD-L1 | MIH1 | m | Acetone | + | 10.00 | affymetrix eBioscience, Santa Clara, CA, USA | ≥1% |
| Positive controls | |||||||
| Epithelial Antigen | Ber-EP4 | m | Acetone | - | 2.50 | Dako, Santa Clara, CA, USA | |
| CD45 | 2B11 + PD7/26 | m | Acetone | - | 4.50 | Dako, Santa Clara, CA, USA | |
| Isotype controls | |||||||
| MOPC 21 | MOPC 21 | m | - | 5.00 | Sigma-Aldrich, St. Louis, MO, USA | ||
| MOPC 21 | m | + | 4.00 | Sigma-Aldrich, St. Louis, MO, USA | |||
| MOPC 21 | m | + | 10.00 | Sigma-Aldrich, St. Louis, MO, USA | |||
| DA1E | DA1E | r | - | 2.12 | Cell Signaling, Danvers, MA, USA | ||
| Biotin conjugated secondary antibodies | |||||||
| 111-065-114 | g anti r | 7.00 | Jackson Immunoresearch, West Grove, PA, USA | ||||
| 315-065-048 | r anti m | 0.75 | Jackson Immunoresearch, West Grove, PA, USA | ||||
Legend: wc: working concentration, m: mouse, r: rabbit, g: goat, all used antibodies’ isotype was IgG1. ERα: estrogen receptor α, PR: progesterone receptor, Her-2/neu: human epidermal growth factor receptor 2, EGFR: epidermal growth factor receptor, HGFR: hepatocyte growth factor receptor, IGF1R: insulin-like growth factor 1, MUC-1: mucin-1, PD-1: programmed cell death protein 1, PD-L1: programmed cell death-ligand 1.